Bt corn flap

[rudy benavides]

Here is another view sent to the MonarchWatch list by Mike Quinn.  I don't 
feel that the complete story on Bt  is in yet.

Rudy Benavides

Volunteer Naturalist
National Wildlife Visitor Center
Laurel, MD

From: Mike Quinn <MQnature at tamu.edu>To: DPLEX-L at UKANS.EDUSubject: Bt Toxin 
persists in soils for at least 234 daysDate: Sun, 26 Mar 2000 22:24:14 -0600


Nature, International Weekly Journal of Science

Nature 402, 480 (1999) (C) Macmillan Publishers Ltd. 02 December 1999

Transgenic plants: Insecticidal toxin in root exudates from Bt corn

DEEPAK SAXENA*, SAUL FLORES* & G. STOTZKY*

* Laboratory of Microbial Ecology, Department of Biology, New York
University, New York, New York 10003, USA
* Instituto Venezolano de Investigaciones Cientificas, Apartado Postal
21827, Caracas 1020A, Venezuela


e-mail: gs5 at is2.nyu.edu


Bt corn is corn (Zea mays) that has been genetically modified to express
insecticidal toxins derived from the bacterium Bacillus thuringiensis to
kill lepidopteran pests feeding on these plants. Here we show that Bt toxin
is released into the rhizosphere soil in root exudates from Bt corn.


The insecticidal toxin produced by B. thuringiensis subsp. kurstaki remains
active in the soil, where it binds rapidly and tightly to clays[1] and humic
acids[2]. The bound toxin retains its insecticidal properties[3] and is
protected against microbial degradation by being bound to soil particles[4],
persisting in various soils for at least 234 days (the longest time
studied), as determined by larvicidal bioassay[5]. Unlike the bacterium, 
which
produces the toxin in a precursor form, Bt corn contains an inserted
truncated cry1Ab gene that encodes the active toxin.

In laboratory studies, caterpillars of the monarch butterfly (Danaus
plexippus) were killed as a result of feeding on milkweed (Asclepias
curassavica) that had been artificially contaminated with pollen from
transgenic corn that expressed the cry1Ab gene from B. thuringiensis subsp.
kurstaki[6], and green lacewings (Chrysoperla carnea), which are insect
predators of insect pests, were killed by ingesting European corn borers
(Ostrinia nubilalis) reared on Bt corn[7].

We germinated surface-sterilized seeds[8] of Bt corn (NK4640Bt) and of the
isogenic strain without the cry1Ab gene on agar. The seedlings were
aseptically placed on plastic screening (6-mm mesh), which, to minimize
contamination by products of endosperm hydrolysis, was suspended over 200 ml
of Hoagland's solution in 4-litre beakers covered with aluminium foil, which
was removed after 18 to 20 days when the tops of the plants had reached it.
After 7, 15 and 25 days of growth in a plant-growth room (2652ƒC, 12 h
light-dark cycle), the soil-free medium was replaced with fresh solution and
analysed.

Total protein in the medium (average of 105 mg protein per plant) was
determined by the Lowry procedure[9]. A major band migrating on SDS-PAGE
(sodium dodecyl sulphate-polyacrylamide gel electrophoresis) to a position
corresponding to a relative molecular mass of 66,000 (66K), the same as that
of the Cry1Ab protein, was evident after 7 and 15 days only in the exudates
from Bt corn, although several protein bands of smaller relative molecular
mass were seen in exudates from both Bt and non-Bt corn. We confirmed the
presence of the toxin in the exudates from Bt corn by immunological assay
with Lateral Flow Quickstix (from EnviroLogix, Maine; detection limit <10
parts per billion) and verified that it was active in an insecticidal
bioassay using larvae of the tobacco hornworm (Manduca sexta), a model for
testing antilepidopteran activity[3, 5].

Larvae placed on medium containing exudates from Bt corn stopped feeding and
began to die after 2 to 3 days and had a mortality of 90 to 95% after 5 days
(dose lethal to 50% of larvae, LC50, was 5.2 ug protein). There was no
immunological reaction or larval mortality obtained with the exudates from
non-Bt corn. After 25 days of growth, when the medium was no longer sterile
(as demonstrated by streaking it on various microbiological media), the 66K
band had disappeared, although there were several new protein bands of
smaller relative molecular mass, and the immunological and larvicidal assays
were negative, indicating that microbial, and probably also corn, proteases
had hydro-lysed the toxin.

Samples of soil from the rhizosphere of seedlings that had been transplanted
into either sterile or non-sterile soil in test-tubes were taken from
randomly selected tubes, vortexed with extraction buffer (EnviroLogix) and
centrifuged. We analysed the supernatants using the immunological and
larvicidal assays and found that these were positive, even after 25 days of
growth, for samples from Bt corn (100% mortality; LC50=1.6 ug protein per
soil tube) but were negative for non-Bt corn. Moreover, particles of
rhizosphere soil in suspension placed directly on the bioassay medium caused
mortality comparable to the supernatants.

Although the concentration of protein in the rhizosphere soil was
approximately 95 g per g soil, the concentration of the actual toxin in the
extraction buffer was apparently too low to be detected by SDS-PAGE. These
results agree with earlier findings showing that the toxin binds rapidly to
surface-active soil particles and that the bound toxin retains its
larvicidal activity and is protected by this binding against
biodegradation[1-5].

About 15 million acres of Bt corn were planted in the United States in 1998,
which was just under 20% of the total acreage of corn[10]. The Bt toxin that
is released into soil from roots during the growth of a Bt corn crop would
add to the amount of toxin introduced into soil from pollen during
tasselling and as a result of the incorporation of plant residues after
harvesting the crop.

We have no indication of how soil communities might be affected by Bt toxin
in root exudates in the field. Bt toxin in the rhizosphere might improve the
control of insect pests, or it might promote the selection of
toxin-resistant target insects. Receptors for the toxin are present in
non-target as well as target insects, so there may be a risk that non-target
insects and organisms in higher trophic levels could be affected by the
toxin. Further investigations will be necessary to shed light on what might
happen underground.


References
  1.     Tapp, H., Calamai, L. & Stotzky, G. Soil Biol. Biochem. 26,
663-679 (1994).
  2.     Crecchio, C. & Stotzky, G. Soil Biol. Biochem. 30, 463-470 (1998).
  3.     Tapp, H. & Stotzky, G. Appl. Environ. Microbiol. 61, 1786-1790
(1995).
  4.     Koskella, J. & Stotzky, G. Appl. Environ. Microbiol. 63, 3561-3568
(1997).
  5.     Tapp, H. & Stotzky, G. Soil Biol. Biochem. 30, 471-476 (1998).
  6.     Losey, J. E., Raynor, L. S. & Carter, M. E. Nature 399, 214 (1999).
  7.     Hilbeck, A., Baumgartner, M., Fried, P. M. & Bigler, F. Environ.
Entomol. 27, 480-487 (1998).
  8.     Benizri, E., Courtade, A. & Guckert, A. Soil Biol. Biochem. 27,
71-77 (1995).
  9.     Lowry, O. H., Rosebough, N. R., Farr, A. L. & Randall, R. J. J.
Biol. Chem. 193, 265-275 (1951).
10.     Wadman, M. Nature 397, 636 (1999).

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